In vitro DNA manipulation and the attendant transfer of genetic information have developed into a technology that allows the efficient expression of endogenous and foreign proteins in microbial hosts. Although expression of any foreign protein in any microbial host is theoretically possible, many problems limit its practice. Stability of the protein produced often limits such practice and results in a low yield. In particular, small foreign proteins and oligopeptides cannot be overproduced in most cellular hosts. While larger proteins can be produced at low or even high yield, their purification from the total cell extract often poses problems.
To address these concerns, several researchers have recently investigated the fusion of a peptide marker to the protein being expressed so that the protein can be readily recognized during purification. For example, a method such as the one described in U.S. Pat. No. 4,782,137, utilizes an antibody immobilized-immunoaffinity technique in combination with an expressed, fused protein containing a small antigenic oligopeptide marker having an "antigenic head" portion and a "linking tail" portion. The antigenic portion is a combination of hydrophilic amino acids that readily elicit antigenic response. The linking portion permits cleavage of the marker from the desired protein after the fused protein has been isolated from the cells.
Other methods involve expression and purification techniques combining the use of enzymatic markers such as glutathione transferase, beta-galactosidase and chloramphenicol acetyl transferase which bind to appropriate substrates. See D. B. Smith, et al., Gene, 67, 31-40 (1988); Japanese application JP62283998, Derwent Abstract No. 4733853 and EPO application 131,363. In these instances, the enzyme is fused to the desired protein, the fusion product separated by binding to an immobilized substrate and the enzyme cleaved from the desired protein by biochemical methods.
Expression of a small polypeptide in a host cell also raises the possibility that the host will assimilate the polypeptide. In instances where the recognition marker and desired protein are small, e.g. below about 60 to 80 amino acid units in length, assimilation rather than expression as an end-product usually occurs.
The efficiency of expression in the host cell can also vary depending upon the character of the protein being produced. The host may produce negligible amounts of fused peptide because the variation of the fused peptide structure can trigger a decrease in the transcriptional and/or translational efficiency. In particular, triggering is a problem with a system that uses a small oligopeptide marker.
Production of fusion proteins containing larger peptides exemplified by beta-galactosidase, glutathione transferase or chloramphenicol acetyl transferase generally creates a new set of difficulties. Purification usually is not very efficient or effective. For such large fusion proteins, the binding constants of the contained enzymes to their substrates usually are so low that purification is difficult to achieve. During purification the substrates often cleave from the column support and contaminate the separated fusion protein. Many of the enzymes are such large molecular weight proteins that the desired protein constitutes only a small fraction of the fusion protein.
Consequently, production and purification of large quantities of protein by a microbial route need to be based upon techniques that avoid or alleviate the foregoing concerns. Overproduction of the desired protein relative to other proteins produced by the host would also be advantageous. The protein used for binding also should be one that has a high affinity for a small molecule, so as to avoid the adverse complexing capacity, leakage, viral contamination and other problems associated with immunoaffinity chromatography.
Therefore, it is an object of the invention to develop an expression system that allows the large scale, high yield overproduction of both small and large polypeptides. A further object is to provide a facile, highly efficient purification scheme that has general applicability to any product polypeptide such as proteins or very small peptides. Yet another object is the development of protein expression and separation methods that utilize inexpensive materials. A further object is practice of the method with soluble affinity systems that permit stable and high yield expression of polypeptides. A particular object is the development of a separation system based upon affinity techniques employing a recombinant and expressed binding protein that has a high affinity for its ligand.